Project Summary Many types of cancer ? such as recurrent glioblastoma multiform, which is invariably lethal ? overexpress bulky glycoproteins to form a thick glycocalyx layer. The glycocalyx physically separates the cell from its surroundings, but recent work has shown that the glycocalyx can paradoxically increase adhesion to soft tissues and therefore promote the metastasis of cancer cells. This surprising phenomenon occurs because the glycocalyx forces adhesion molecules (called integrins) on the cell's surface into clusters. These integrin clusters have cooperative effects that allow them to form stronger adhesions to surrounding tissues than would be possible with equivalent numbers of un-clustered integrins. These cooperative mechanisms have been intensely scrutinized in recent years; a more nuanced understanding of the biophysical underpinnings of glycocalyx- mediated adhesion could uncover therapeutic targets, deepen our general understanding of cancer metastasis, and elucidate general biophysical processes that extend far beyond the realm of cancer research. Here I present the hypothesis that the glycocalyx has the additional effect of increasing mechanical tension experienced by clustered integrins. Integrins function as mechanosensors that undergo structure-switching into ?active? conformations when subjected to mechanical tension. As such, my hypothesis would, if true, suggest a more immediate regulatory role of the glycocalyx in adhesion than previously realized. This hypothesis (which I call the local organization hypothesis) is well-supported through indirect observation in the research literature but has not been directly tested due to challenges associated with measuring mechanical tension on individual biomolecules in live cells. However, I have devoted much of my career to developing DNA-based mechanosensor tools that can be used to directly measure piconewton-scale integrin tension in live cells. Here I propose to utilize these tools to test the local organization hypothesis and bring clarity to this rapidly-growing research field. I plan to image integrin forces during the early stages of cellular adhesion and test for a set of specific observations that would support or refute the local organization hypothesis. In parallel, I plan to leverage my computational skills to test this hypothesis using a sophisticated chemomechanical simulation method. For my postdoctoral (K00) work, I will transition into translational work and develop tools that leverage the principles of glycocalyx-mediated adhesion for cancer diagnostic purposes. Because glycocalyx-presenting cancer cells adhere more readily to soft substrates, I will develop a flow-based method for detecting circulating tumor cells (CTCs) using soft substrates and substrates of varying stiffness that facilitate mechanoselection of glycocalyx-presenting cancer cells. This mechanoselection-based method will enable mechanical profiling of CTCs to determine what types of tissues are most vulnerable to metastasis and will also allow for conventional biochemical profiling in parallel. This work will deliver a substantially enhanced understanding of the mechanisms of metastasis, as well as tools that can put this improved understanding to diagnostic use in clinical settings.